And Calcium-Dependent Vascular Effects of Polybia Paulista Wasp Er
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The Journal of Venomous Animals and Toxins including Tropical Diseases ISSN 1678-9199 | 2011 | volume 17 | issue 2 | pages 199-208 Renal- and calcium-dependent vascular effects of Polybia paulista wasp ER P venom A P Vinhote JFC (1), Torres AFC (2), Dantas RT (1), Praciano TP (2), Menezes RRPPB (1), Sousa DF (1), Brito RIGINAL TS (1), Lima FJB (1), Toyama MH (3), Magalhães PJ (1), Monteiro HSA (1), Martins-Nunes AMC (2) O (1) Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Ceará State, Brazil; (2) Department of Clinical and Toxicological Analyses, Federal University of Ceará, Fortaleza, Ceará State, Brasil; (3) São Paulo Experimental Coast Campus, São Paulo State University (UNESP – Univ Estadual Paulista), São Vicente, São Paulo State, Brazil. Abstract: In the present study, the effects of Polybia paulista venom (PPV) on renal and vascular tissues were investigated. Isolated kidneys perfused with PPV (1 and 3 µg/mL) had increased perfusion pressure, renal vascular resistance, urinary flow, and glomerular filtration rate; and reduced sodium tubular transport. Histological evaluation demonstrated deposits of proteins in Bowman’s space and tubular lumen, and focal areas of necrosis. The venom promoted a cytotoxic effect on Madin-Darby canine kidney (MDCK) cells. A significant increase in lactic dehydrogenase levels was observed in response to venom exposure. In isolated mesenteric vascular beds, pressure and vascular resistance augmented in a dose-dependent manner. PPV increased the contractility of aortic rings maintained under basal tension. This contractile response was inhibited when preparations were maintained in Ca2+-free medium. Likewise, verapamil, a voltage-gated calcium channel blocker, also inhibited the contractile response. In this study, phentolamine, a blocker of α-adrenergic receptor blocker, significantly reduced the contractile effect of PPV in the aortic ring. In conclusion, PPV produced nephrotoxicity, which suggests a direct effect on necrotic cellular death in renal tubule cells. The vascular contractile effect of PPV appears to involve calcium influx through voltage-gated calcium channels via adrenergic regulation. Key words: Polybia paulista, venom, kidney, aorta, MDCK. INTRODUCTION nucleases, histamine and degranulating peptide mastoparan – are also reported to be components In Brazil, there are several species of social of Vespinae venoms, and have direct and indirect wasps and little is known about the toxicological cytotoxic, hemolytic, neurotoxic and vasoactive properties of their venoms. Many of these species, properties (3-6). such as the neotropical wasp Polybia paulista, are Incidents involving these insects can be involved in stinging incidents in urban areas in harmful, with a large number of patients, southeastern Brazil (1). The victims commonly mainly allergic individuals, developing severe describe severe pain and local inflammation, complications, including acute renal failure (ARF) symptoms that reveal intense biological activity (7, 8). It has been postulated that the underlying of P. paulista (PPV). Recently, a study reported mechanism of ARF involves a direct nephrotoxic hyperalgesic and edematogenic effects of peptides effect following wasp poisoning, which may then isolated from PPV (2). The constituents of PPV result in tubulointerstitial nephritis and acute – such as melittin, apamin, phospholipases, tubular necrosis, possibly in association with hyaluronidases, acid phosphatases, proteases, hemolysis or rhabdomyolysis (9-14). In fact, Vinhote JFC, et al. Renal and vascular effects of Polybia paulista wasp venom mastoparans, polycationic peptides isolated from of bovine serum albumin (BSA) was added to PPV, cause apoptosis involving caspase-signaling 100 mL of MKHS and dialyzed for 48 hours at pathways with mitochondrial damage, and 4°C against 10 volumes of MKHS. Immediately cytokine activation (15). before the beginning of each perfusion protocol, Despite the demonstration of the myotoxic 100 mg of urea, 50 mg of inulin and 50 mg of effects of its constituents, the deleterious effects of glucose were added to every 100 mL of perfusate, PPV on kidneys have not yet been defined. Thus, and the pH was adjusted to 7.4. the present study was designed to characterize In each experiment, 100 mL of MKHS was the effects of PPV on renal and vascular tissues. recirculated for 120 minutes. Perfusion pressure (PP) was measured at the tip of a stainless steel MATERIAL AND METHODS cannula in the renal artery. Samples of urine and perfusate were collected at 10-minute intervals Animals for the determination of sodium, chloride and Male Wistar rats (250-350 g) were maintained potassium levels by means of ion-selective under standard conditions of temperature and electrodes (Rapid Chem 744®, Bayer Diagnostic, humidity in 12-hour light-dark cycles. The UK). Inulin was determined by direct hydrolysis, animals were fasted for 24 hours before any as described by Walser et al. (18) and modified experimental procedure and water was provided by Fonteles et al. (17). Osmolality was measured ad libitum. All experiments were in accordance using a vapor pressure osmometer (5100C, with the guidelines for the ethical use of Wescor, USA). PPV (1 or 3 mg/mL) was added to experimental animals published by the Brazilian the system just after the basal perfusion pressure College on Experimental Animal Care (COBEA), had stabilized (30 minutes after the beginning of and experimental procedures were approved each experiment). Perfusion pressure (PP), renal by the Institutional Animal Care Committee at vascular resistance (RVR), urinary flow (UF), and Federal University of Ceará (protocol number glomerular filtration rate (GFR) were evaluated. 17/09). Urine and perfusate samples were collected at 30-minute intervals to evaluate the percentage Venom and Reagents tubular transport for Na+ (% TNa+), K+ (% TK+) Polybia paulista wasp venom was kindly and Cl– (% TCl–) as determined by Pitts (19) and provided by Prof. M. H. Toyama of São Paulo State Martinez-Maldonato and Opava-Stitzer (20). University (UNESP). All chemicals and analytical grade solvents were purchased from commercially Histological evaluation available suppliers (Bayer, Sigma-Aldrich, Labtest After completion of the experiment, the Diagnostic and Applied Biosystems). kidney was removed from the experimental set- up and kept in 10% formaldehyde for histological Renal Effects Induced by PPV evaluation. Hematoxylin and eosin staining were used for light microscopy analysis. Isolated rat kidney assay Rat kidneys were isolated and perfused Cytotoxic effects induced by PPV according to the method described by Bowman Epithelial Madin-Darby canine kidney (16) and modified by Fonteles et al. (17). Briefly, (MDCK) cells were cultured in RPMI-1640 rats were anesthetized with sodium pentobarbital medium, supplemented with FBS (10%), (50 mg/kg, intraperitoneally). The abdomen was penicillin (100 U/mL), and streptomycin (100 opened through a midline incision, and the right µg/mL). Cells were seeded at a concentration of kidney was exposed. After careful dissection 1 x 105 cells/mL in microplates, and incubated at of the kidney, the renal artery was cannulated 37°C with 5% CO2 for two hours. After cells were via the mesenteric artery without interrupting washed with sterile phosphate buffered solution blood flow. The perfusate solution consisted of (PBS) at pH 7.4, they were treated with PPV (3.12, a modified Krebs-Henseleit solution (MKHS) 6.25, 12.5, 25, 50 or 100 µg/mL) for 24 hours and with the following composition in mmol/L: 118.0 then evaluated using an inverted microscope. NaCl, 1.2 KCl, 1.18 KH2PO4, 1.18 MgSO4.7H2O, The negative control was prepared with medium 2.50 CaCl2.7H2O and 25.0 NaHCO3. Six grams plus cells and PBS instead of venom. Before each J Venom Anim Toxins incl Trop Dis | 2011 | volume 17 | issue 2 200 Vinhote JFC, et al. Renal and vascular effects of Polybia paulista wasp venom experiment, cells were stored in medium without artery were tied. Then, the superior mesenteric FBS for 24 hours to obtain cells in phase G0 of the artery was cleaned of surrounding tissue and cell cycle (21). cannulated with a polyethylene tube (PE20). The intestine was separated from the mesenteric bed MTT assay by cutting close to the intestinal border of the MDCK cells were cultured as previously mesentery. The mesenteric bed was perfused with described, treated with PPV or PBS (negative Krebs solution containing: NaCl 114.0 mM; KCl control) and subjected to the MTT assay in 4.96 mM; KH2PO4 1.24 mM; MgSO4.7H2O 0.5 microtiter plates after 24 hours of incubation. mM; NaHCO3 24.99 mM; CaCl2.2H2O 2.10 mM; The supernatant of the cells was removed and glucose 3.60 mM. The perfusion solution and 3-(4,5-dimethythiazol-2-yl)-2,5 was kept warm at 37°C, and the mesenteric bed diphenyltetrazolium (MTT) (2.5 mg/mL in PBS; was perfused using a constant flow rate (4 mL/ Sigma, Brazil) was added to each plate well (10 minute). µL/well). The method is based on reduction of Changes in perfusion pressure were tetrazolium salt by active mitochondria in living continuously measured with a pressure transducer cells to insoluble purple formazan crystals (22). (Statham P23, Gould, USA) and recorded using After incubation for four hours at 37°C with 5% a four-channel physiograph (Narco BioSystems, CO2, the supernatant was removed, and 10% USA). After the basal perfusion pressure had sodium dodecyl sulfate (SDS) in 0.01N HCL stabilized, the mesenteric vascular beds were was added to solubilize the formazan crystals. perfused with Krebs solution containing PPV (3, The plates were incubated for 17 hours, and then 10 or 100 mg/mL; n = 6) for ten minutes. Changes absorbance was measured by spectrophotometry in the perfusion pressure of the mesenteric bed (570 nm). Cell viability was determined by induced by PPV were compared to perfusion evaluating the mean percentage of surviving cells pressure recorded when preparations were at a given PPV concentration. Survival measured perfused with vehicle alone at the same rate. in the negative control was taken to be equal to Perfusion of the mesenteric bed with a solution of 100%.